Long-term biodegradation of crude oil in high-arctic backshore sediments: The Baffin Island Oil Spill (BIOS) after nearly four decades.

With an on-going disproportional warming of the Arctic Ocean and the reduction of the sea ice cover, the risk of an accidental oil spill from ships or future oil exploration is increasing. It is hence important to know how crude oil weathers in this environment and what factors affect oil biodegradation in the Arctic. However, this topic is currently poorly studied. In the 1980s, the Baffin Island Oil Spill (BIOS) project carried out a series of simulated oil spills in the backshore zone of beaches located on Baffin Island in the Canadian High Arctic. In this study two BIOS sites were re-visited, offering the unique opportunity to study the long-term weathering of crude oil under Arctic conditions. Here we show that residual oil remains present at these sites even after almost four decades since the original oiling. Oil at both BIOS sites appears to have attenuated very slowly with estimated loss rates of 1.8-2.7% per year. The presence of residual oil continues to significantly affect sediment microbial communities at the sites as manifested by a significantly decreased diversity, differences in the abundance of microorganisms and an enrichment of putative oil-degrading bacteria in oiled sediments. Reconstructed genomes of putative oil degraders suggest that only a subset is specifically adapted for growth under psychrothermic conditions, further reducing the time for biodegradation during the already short Arctic summers. Altogether, this study shows that crude oil spilled in the Arctic can persist and significantly affect the Arctic ecosystem for a long time, in the order of several decades.

Biodegradation potential of residue generated during the in-situ burning of oil in the marine environment.

The biodegradability of residues derived from in-situ burning, an oil spill response strategy which involves burning an oil slick on the sea surface, has not yet been fully studied. With a growing risk of oil spills, the fate of the persistent burn residue containing potentially toxic substances must be better understood. Microcosms were used to study the microbial community response and potential biodegradability of in-situ burn residues generated from Ultra Low Sulphur (ULS) marine diesel. Microcosm studies were conducted using residues originating from the burning of unweathered and weathered diesel, with the addition of a fertilizer and a dispersant. Burn residues were incubated for 6 weeks at 7 °C in natural seawater with continual agitation in the dark. Samples were subsequently sacrificed for chemistry as well as 16S rRNA gene amplicon and shotgun metagenomic sequencing. Chemistry analyses revealed a reduction in hydrocarbon concentrations. Medium chain-length n-alkanes (nC16-nC24) decreased by 8% in unweathered burn residue microcosms and up to 26% in weathered burn residue microcosms. A significant decrease in polycyclic aromatic hydrocarbon (PAH) concentrations was observed only for naphthalene, fluorene and their alkylated homologs, in the microcosms amended with residue produced from burning weathered diesel. Decreases of 2-24%, were identified depending on the compound. Microcosms amended with burn residues had distinct microbial communities marked by an increase in relative abundance of putative hydrocarbon degraders as well as an increase of known hydrocarbon-degradation genes. These novel results suggest that if in-situ burning is performed on ULS marine diesel, some of the indigenous bacteria would respond to the newly available carbon source and some of the residual compounds would be biodegraded. Future studies involving longer incubation periods could give a better understanding of the fate of burn residues by shedding light on the potential biodegradability of the more recalcitrant residual compounds.

An experimental oil spill at a tidal freshwater wetland along the St. Lawrence River re-visited after 21 years.

In 1999, a tidal wetland located along the St. Lawrence River close to Ste. Croix de Lotbinière (Quebec, Eastern Canada) was the site of an experimental oil spill. Test plots were established and subjected to an experimental crude oil spill to evaluate natural attenuation, nutrient amendment and vegetation cropping as countermeasures. In 2020, this study re-visited the test plots to investigate residual oil and habitat recovery. Only concentrations of mid-chain length n-alkanes (C10-C36), but not of polycyclic aromatic hydrocarbons (PAHs), were significantly above detection limit, and were detected in both test plot and control sediments. Hydrocarbon, total organic carbon, nitrogen and phosphate contents did not differ significantly between test plot and control sediments. Microbial analyses did not detect significant differences in microbial load, microbial diversity or microbial community composition between test plot and control sediments. Key genes for the aerobic and anaerobic degradation of n-alkanes as well as for the aerobic degradation of PAHs were detected in all sediment samples. Associated gene abundances did not differ significantly between test plot and control sediments. This study shows that oil-exposed test plot sediments of the Ste. Croix wetland can be considered completely recovered after 21 years irrespective of the performed countermeasure.

Metagenomic and Metatranscriptomic Responses of Chemical Dispersant Application during a Marine Dilbit Spill.

The global increase in marine transportation of dilbit (diluted bitumen) can increase the risk of spills, and the application of chemical dispersants remains a common response practice in spill events. To reliably evaluate dispersant effects on dilbit biodegradation over time, we set large-scale (1,500 mL) microcosms without nutrient addition using a low dilbit concentration (30 ppm). Shotgun metagenomics and metatranscriptomics were deployed to investigate microbial community responses to naturally and chemically dispersed dilbit. We found that the large-scale microcosms could produce more reproducible community trajectories than small-scale (250 mL) ones based on the 16S rRNA gene amplicon sequencing. In the early-stage large-scale microcosms, multiple genera were involved in the biodegradation of dilbit, while dispersant addition enriched primarily Alteromonas and competed for the utilization of dilbit, causing depressed degradation of aromatics. The metatranscriptomic-based metagenome-assembled genomes (MAG) further elucidated early-stage microbial antioxidation mechanism, which showed that dispersant addition triggered the increased expression of the antioxidation process genes of Alteromonas species. Differently, in the late stage, the microbial communities showed high diversity and richness and similar compositions and metabolic functions regardless of dispersant addition, indicating that the biotransformation of remaining compounds can occur within the post-oil communities. These findings can guide future microcosm studies and the application of chemical dispersants for responding to a marine dilbit spill. IMPORTANCE In this study, we employed microcosms to study the effects of marine dilbit spill and dispersant application on microbial community dynamics over time. We evaluated the impacts of microcosm scale and found that increasing the scale is beneficial for reducing community stochasticity, especially in the late stage of biodegradation. We observed that dispersant application suppressed aromatics biodegradation in the early stage (6 days), whereas exerting insignificant effects in the late stage (50 days), from both substance removal and metagenomic/metatranscriptomic perspectives. We further found that Alteromonas species are vital for the early-stage chemically dispersed oil biodegradation and clarified their degradation and antioxidation mechanisms. These findings help us to better understand microcosm studies and microbial roles for biodegrading dilbit and chemically dispersed dilbit and suggest that dispersant evaluation in large-scale systems and even through field trails would be more realistic after marine oil spill response.

Biodegradation of weathered crude oil in seawater with frazil ice.

As ice extent in the Arctic is declining, oil and gas activities will increase, with higher risk of oil spills to the marine environment. To determine biotransformation of dispersed weathered oil in newly formed ice, oil dispersions (2-3 ppm) were incubated in a mixture of natural seawater and frazil ice for 125 days at -2 °C. Dispersed oil in seawater without frazil ice were included in the experimental setup. Presence or absence of frazil ice was a strong driver for microbial community structures and affected the rate of oil degradation. n-alkanes were degraded faster in the presence of frazil ice, the opposite was the case for naphthalenes and 2-3 ring PAHs. No degradation of 4-6 ring PAHs was observed in any of the treatments. The total petroleum oil was not degraded to any significant degree, suggesting that oil will freeze into the ice matrix and persist throughout the icy season.

Long-term effects of nitrogen and phosphorus fertilization on soil microbial community structure and function under continuous wheat production.

Soil microorganisms play a critical role in the biosphere, and the influence of cropland fertilization on the evolution of soil as a living entity is being actively documented. In this study, we used a shotgun metagenomics approach to globally expose the effects of 50-year N and P fertilization of wheat on soil microbial community structure and function, and their potential involvement in overall N cycling. Nitrogen (N) fertilization increased alpha diversity in archaea and fungi while reducing it in bacteria. Beta diversity of archaea, bacteria and fungi, as well as soil function, were also mainly driven by N fertilization. The abundance of archaea was negatively impacted by N fertilization while bacterial and fungal abundance was increased. The responses of N metabolism-related genes to fertilization differed in archaea, bacteria and fungi. All archaeal N metabolic processes were decreased by N fertilization, while denitrification, assimilatory nitrate reduction and organic-N metabolism were highly increased by N fertilization in bacteria. Nitrate assimilation was the main contribution of fungi to N cycling. Thaumarchaeota and Halobacteria in archaea; Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria in bacteria; and Sordariomycetes in fungi participated dominantly and widely in soil N metabolic processes.

Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Nitrogen- and phosphorus-starved Triticum aestivum show distinct belowground microbiome profiles.

Many plants have natural partnerships with microbes that can boost their nitrogen (N) and/or phosphorus (P) acquisition. To assess whether wheat may have undiscovered associations of these types, we tested if N/P-starved Triticum aestivum show microbiome profiles that are simultaneously different from those of N/P-amended plants and those of their own bulk soils. The bacterial and fungal communities of root, rhizosphere, and bulk soil samples from the Historical Dryland Plots (Lethbridge, Canada), which hold T. aestivum that is grown both under N/P fertilization and in conditions of extreme N/P-starvation, were taxonomically described and compared (bacterial 16S rRNA genes and fungal Internal Transcribed Spacers-ITS). As the list may include novel N- and/or P-providing wheat partners, we then identified all the operational taxonomic units (OTUs) that were proportionally enriched in one or more of the nutrient starvation- and plant-specific communities. These analyses revealed: a) distinct N-starvation root and rhizosphere bacterial communities that were proportionally enriched, among others, in OTUs belonging to families Enterobacteriaceae, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Cytophagaceae, Streptomycetaceae, b) distinct N-starvation root fungal communities that were proportionally enriched in OTUs belonging to taxa Lulworthia, Sordariomycetes, Apodus, Conocybe, Ascomycota, Crocicreas, c) a distinct P-starvation rhizosphere bacterial community that was proportionally enriched in an OTU belonging to genus Agrobacterium, and d) a distinct P-starvation root fungal community that was proportionally enriched in OTUs belonging to genera Parastagonospora and Phaeosphaeriopsis. Our study might have exposed wheat-microbe connections that can form the basis of novel complementary yield-boosting tools.

Soil contamination alters the willow root and rhizosphere metatranscriptome and the root-rhizosphere interactome.

Phytoremediation using willows is thought to be a sustainable alternative to traditional remediation techniques involving excavation, transport, and landfilling. However, the complexity of the interaction between the willow and its associated highly diverse microbial communities makes the optimization of phytoremediation very difficult. Here, we have sequenced the rhizosphere metatranscriptome of four willow species and the plant root metatranscriptome for two willow species growing in petroleum hydrocarbon-contaminated and non-contaminated soils on a former petroleum refinery site. Significant differences in the abundance of transcripts related to different bacterial and fungal taxa were observed between willow species, mostly in contaminated soils. When comparing transcript abundance in contaminated vs. non-contaminated soil for each willow species individually, transcripts for many microbial taxa and functions were significantly more abundant in contaminated rhizosphere soil for Salix eriocephala, S. miyabeana and S. purpurea, in contrast to what was observed in the rhizosphere of S. caprea. This agrees with the previously reported sensitivity of S. caprea to contamination, and the superior tolerance of S. miyabeana and S. purpurea to soil contamination at that site. The root metatranscriptomes of two species were compared and revealed that plants transcripts are mainly influenced by willow species, while microbial transcripts mainly responded to contamination. A comparison of the rhizosphere and root metatranscriptomes in the S. purpurea species revealed a complete reorganization of the linkages between root and rhizosphere pathways when comparing willows growing in contaminated and non-contaminated soils, mainly because of large shifts in the rhizosphere metatranscriptome.

Chemical dispersants enhance the activity of oil- and gas condensate-degrading marine bacteria.

Application of chemical dispersants to oil spills in the marine environment is a common practice to disperse oil into the water column and stimulate oil biodegradation by increasing its bioavailability to indigenous bacteria capable of naturally metabolizing hydrocarbons. In the context of a spill event, the biodegradation of crude oil and gas condensate off eastern Canada is an essential component of a response strategy. In laboratory experiments, we simulated conditions similar to an oil spill with and without the addition of chemical dispersant under both winter and summer conditions and evaluated the natural attenuation potential for hydrocarbons in near-surface sea water from the vicinity of crude oil and natural gas production facilities off eastern Canada. Chemical analyses were performed to determine hydrocarbon degradation rates, and metagenome binning combined with metatranscriptomics was used to reconstruct abundant bacterial genomes and estimate their oil degradation gene abundance and activity. Our results show important and rapid structural shifts in microbial populations in all three different oil production sites examined following exposure to oil, oil with dispersant and dispersant alone. We found that the addition of dispersant to crude oil enhanced oil degradation rates and favored the abundance and expression of oil-degrading genes from a Thalassolituus sp. (that is, metagenome bin) that harbors multiple alkane hydroxylase (alkB) gene copies. We propose that this member of the Oceanospirillales group would be an important oil degrader when oil spills are treated with dispersant.